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The Journal of Neuroscience, September 1, 2002, 22(17):7627-7638
Distinct Actions of Emx1, Emx2, and
Pax6 in Regulating the Specification of Areas in the
Developing Neocortex
Kathie M.
Bishop1,
John
L. R.
Rubenstein2, and
Dennis D. M.
O'Leary1
1 Molecular Neurobiology Laboratory, The Salk Institute
for Biological Studies, La Jolla, California 92037, and
2 Nina Ireland Laboratory of Developmental Neurobiology,
Department of Psychiatry, University of California San Francisco, San
Francisco, California 94143
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ABSTRACT |
The mammalian neocortex is organized into subdivisions referred to
as areas that are distinguished from one another by differences in
architecture, axonal connections, and function. The transcription factors EMX1, EMX2, and PAX6 have been proposed to regulate
arealization. Emx1 and Emx2 are expressed
by progenitor cells in a low rostrolateral to high caudomedial
gradient across the embryonic neocortex, and Pax6 is
expressed in a high rostrolateral to low caudomedial gradient. Recent
evidence has suggested that EMX2 and PAX6 have a role in the genetic
regulation of arealization. Here we use a panel of seven genes
(Cad6, Cad8, Id2,
RZR
, p75, EphA7,
and ephrin-A5) representative of a broad range of
proteins as complementary markers of positional identity to obtain a
more thorough assessment of the suggested roles for EMX2 and PAX6 in
arealization, and in addition to assess the proposed but untested role
for EMX1 in arealization. Orderly changes in the size and positioning
of domains of marker expression in Emx2 and
Pax6 mutants strongly imply that rostrolateral areas
(motor and somatosensory) are expanded, whereas caudomedial areas
(visual) are reduced in Emx2 mutants and that opposite
effects occur in Pax6 mutants, consistent with their opposing gradients of expression. In contrast, patterns of marker expression, as well as the distribution of area-specific
thalamocortical projections, appear normal in Emx1
mutants, indicating that they do not exhibit changes in arealization.
This lack of a defined role for EMX1 in arealization is supported by
our finding of similar shifts in patterns of marker expression in
Emx1; Emx2 double mutants as in Emx2 mutants.
Thus, our findings indicate that EMX2 and PAX6 regulate, in opposing
manners, arealization of the neocortex and impart positional identity
to cortical cells, whereas EMX1 appears not to have a role in this process.
Key words:
area identity; cadherins; corticogenesis; EphA7; ephrin-A5; Id2; p75; patterning mechanisms; positional identity; ROR; RZR; thalamocortical projections; transcription
factors
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INTRODUCTION |
The neocortex, a dorsal
telencephalic structure unique to mammals, is the largest region of the
cerebral cortex and is responsible for sensory perception, cognition,
and volitional control of movements. In its tangential dimension, the
neocortex is organized into subdivisions referred to as areas. Areas
are distinguished from one another by major differences in their
architecture and axonal connections, which in large part determine the
functional specializations that characterize areas in the adult. It has
been assumed that the specification and differentiation of neocortical
areas during development are controlled by an interplay between genetic
regulation intrinsic to the neocortex and extrinsic influences arising
from outside the neocortex (Rakic, 1988
; O'Leary, 1989).
Until recently, most experimental evidence has implicated extrinsic
mechanisms in arealization, in particular the influences of
thalamocortical axons (TCAs), the major afferent projections to the
cortex that define the modality of the primary sensory areas (Chenn et
al., 1997). Evidence for the genetic regulation of arealization has
only begun to emerge over the last 3 years (Monuki and Walsh, 2001;
Ragsdale and Grove, 2001; O'Leary and Nakagawa, 2002). The initial
evidence was indirect and limited to descriptions of graded or
restricted patterns of gene expression across the ventricular zone or
the cortical plate before TCAs enter the neocortex (Donoghue and
Rakic, 1999; Mackarehtschian et al., 1999; Miyashita-Lin et al., 1999;
Nakagawa et al., 1999). Analyses of Gbx2 and
Mash1 mutant mice, which fail to develop a TCA projection
(Miyashita-Lin et al., 1999; Tuttle et al., 1999), have shown that
these differential patterns of gene expression are established and
maintained in the embryonic neocortex independent of TCA input
(Miyashita-Lin et al., 1999; Nakagawa et al., 1999) and are therefore
likely controlled by mechanisms intrinsic to the dorsal telencephalon.
The closely related homeodomain transcription factors EMX1 and EMX2,
and the paired-box domain transcription factor PAX6, have been
proposed to be genetic regulators of arealization (O'Leary et al.,
1994). Emx1 and Emx2 are expressed in a low
rostrolateral to high caudomedial gradient (Gulisano et al., 1996;
Mallamaci et al., 1998) and Pax6 in a high rostrolateral to
low caudomedial gradient (Stoykova and Gruss, 1994) across the
embryonic neocortex. The expression of Emx2 and PAX6 is
restricted primarily to cortical progenitors cells, whereas
Emx1 is expressed in both progenitors and their postmitotic
progeny. If involved in arealization, EMX1 and EMX2 should
preferentially impart caudal and medial area identities (such as
visual), whereas PAX6 should preferentially impart rostral and
lateral area identities (such as motor and somatosensory).
Recent studies have presented evidence for a role for EMX2 (Bishop
et al., 2000; Mallamaci et al., 2000a) and PAX6 (Bishop et al.,
2000) in arealization by analyzing Emx2 and Pax6
Small eye (Sey/Sey) mutant
mice. Because Emx2 and Pax6 mutants die on the
day of birth, before areas become anatomically and functionally distinct, these analyses relied mainly on the use of genes expressed in
differential patterns across the neocortex as putative molecular markers of area identity. Changes in marker expression and patterns of
area-specific TCA projections suggested that rostrolateral areas are
expanded, whereas caudomedial areas are reduced in Emx2 mutants. The analysis of the Pax6 mutants was limited to the
patterned expression of Cad6 and Cad8 (Bishop et
al., 2000), which exhibit changes opposite to those in Emx2
mutants, suggesting that rostrolateral areas are reduced and
caudomedial areas are expanded in the Pax6 mutants.
Because the interpretations in these studies were based on very limited
markers, not only in number but also in the classes of genes
represented and in their patterns of expression, we have performed a
more thorough marker analysis of arealization in Emx2 and
Pax6 mutants, using a battery of seven complementary markers representative of a broad range of genes. These genes encode a broad
range of proteins, including cell adhesion molecules (Cad6 and Cad8), an HLH transcription factor
(Id2), an orphan nuclear receptor (RZR, also
termed ROR), and a neurotrophin receptor (p75), as well as receptors and ligands involved in
axon guidance and cell migration (EphA7 and
ephrin-A5). This more extensive analysis is especially
critical for substantiating the role of PAX6 in arealization, because
Pax6 mutants lack a TCA projection, and therefore the
area-specific patterning of this projection cannot be studied. In
addition, we analyze sectioned material that reveals more complex and
informative expression patterns than observed in the previous
whole-mount in situ analysis.
The similarities in the graded expression of Emx1 and
Emx2, and their close sequence homologies, have prompted the
proposal that EMX1 also may regulate arealization of the neocortex
(Cecchi and Boncinelli, 2000). Therefore, we have also addressed its
role in arealization by analyzing in Emx1 mutant mice the
patterned expression of the set of seven gene markers, as well as
area-specific TCA projections. To our surprise, the Emx1
mutants do not exhibit defects in arealization. To assess whether this
may be attributable to Emx2 compensating for the loss of
Emx1, we performed a marker analysis of Emx1;
Emx2 double mutants. Our findings indicate that EMX2 and
PAX6 disproportionately regulate arealization of the neocortex in
opposing manners, whereas EMX1 has no apparent role in this process.
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MATERIALS AND METHODS |
Mice. Single-mutant Emx1 and
Emx2 mice used for this study were generated from
heterozygous breeding pairs, which allowed for a direct comparison of
gene expression patterns between +/+, +/
, and / littermates for
each mutant studied. Emx2 mice were maintained on a C57/BL6
background, and Emx1 mice were maintained on a 129/Sv
background. To generate Emx1; Emx2 double mutant embryos, Emx1+/ mice were crossed with Emx2+/ mice.
Offspring were genotyped and Emx1+/; Emx2+/ and
Emx1/; Emx2+/ mice were bred with each other to obtain
double mutant embryos and their littermates. Emx2
(Pelligrini et al., 1996), Emx1 (Qiu et al., 1996), and
Emx1; Emx2 double mutant mice were genotyped by
PCR as described. PAX6 mutants were obtained from mating
heterozygous Sey mice (Hill et al., 1991) maintained on a
C57BL/6J × DBA/2J background. Sey mice were genotyped
by eye morphology: Sey/Sey mice lack eyes, and
+/Sey mice have a pronounced reduction in the external size of the eye and the lens at embryonic day (E) 18.5 (Hill et al., 1991).
Because Emx2/, Sey/Sey, and Emx1/;
Emx2/ mice die within a few hours after birth, embryos were
removed and fixed at E18.5, just before birth, which for these mouse
strains occurs at approximately E19.0. Although Emx1 null
mice live to adulthood and are fertile (Qiu et al., 1996), we used
newborn [postnatal day (P) 0] Emx1 mice to facilitate
comparisons of gene expression patterns between the three mutants
analyzed in this study. Analyses were done blinded to genotype. Midday
of the day of vaginal plug detection was considered E0.5, and the first
24 hr after birth is termed P0. Animal care was in accordance with
institutional guidelines.
In situ hybridization. For in situ
hybridization on sections, brains were fixed with 4% paraformaldehyde
(PFA) in 0.1 M phosphate buffer (PB),
cryoprotected with 30% sucrose in PFA/PB, and cut at 20 µm in the
sagittal plane on a cryostat. In situ hybridization using
35S-labeled riboprobes and counterstaining
with bisbenzimide were performed as described previously (Liu et al.,
2000). The following riboprobes were synthesized from cDNA templates:
Cad6 (from S. Mah and C. Kintner); Cad8;
Id2 (from M. Israel), RZR (from M. Becker-Andre, Serono Pharmaceutical Research Institute,
Plan-Les-Ouates, Switzerland), p75 (from K.-F. Lee, Salk
Institute, LaJolla, CA), EphA7 (from A. Brown and G. Lemke,
Salk Institute, LaJolla, CA) and ephrin-A5.
Whole-mount in situ hybridization of the intact forebrain
and midbrain was performed with digoxygenin-labeled riboprobes for
Cad6 and Cad8 using a modification of the
protocol of Henrique et al. (1995), described in Nakagawa et al.
(1999).
Retrograde axon labeling. Newborn mice were perfused
transcardially with 4% PFA in PB, and their brains were removed and
postfixed overnight. Thalamocortical projection neurons were
retrogradely labeled using crystals of the lipophilic dyes
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
(DiI) (Molecular Probes, Eugene, OR) and
4-[4-(dihexadecylamino)stryryl]-N-methylpyridinium iodide (DiA) (Molecular Probes) (Honig and Hume, 1989a,b) placed into the neocortex of Emx1+/+, +/, and / littermates.
In the same cortical hemisphere, a single small crystal of DiI was
placed at a site that in a wild-type mouse would be near the center of primary visual area, and a single small crystal of DiA was placed at a
site that in a wild-type mouse would be near the center of the primary
somatosensory area. Care was taken to equate crystal size and placement
between littermate sets of brains. In addition, we attempted to place
the DiA crystal in the cortical plate and not involve the underlying
subplate, the intracortical pathway of TCAs, to avoid the possible
labeling of dorsal lateral geniculate thalamic nucleus (dLG) axons en
route to the primary visual area. Brains were stored for 2 weeks
at 30°C in fixative, which was sufficient time to completely fill
thalamocortical projection neurons. The brains were then cut sagitally
at 100 µm on a vibratome. Sections were counterstained with
bisbenzimide, and every section through the thalamus and cortical
crystal sites was serially mounted. Sections were analyzed and
photographed with a Nikon upright fluorescence microscope using RITC
(DiI), FITC (DiA) and UV (bisbenzimide) filter cubes. Before
sectioning, the dorsal surface of the cortical hemisphere was
photographed to document the locations of the DiI and DiA crystal
placements; the placement sites and their sizes were further documented
in the sections.
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RESULTS |
In the major component of this study, we examined in wild-type
mice, in Pax6, Emx2 and Emx1 mutant
mice, and in Emx1; Emx2 double mutant mice, the expression
of seven genes (Cad8, Cad6, Id2,
p75, RZR, ephrin-A5, and
EphA7) that in wild-type mice exhibit restricted or
differential patterns of expression across the embryonic neocortex (see
introductory remarks). Therefore, these genes are useful as molecular
markers of position-dependent or areal properties of neocortical cells.
Our analyses were done on E18.5 (hours before birth) or P0, the day
Emx2 and Pax6 mutants die. Figure
1 presents in a schematic manner the
graded expression of Emx1, Emx2, and Pax6 and summarizes our predictions and findings from
loss-of-function mutant analyses. For simplicity, we use the terms
motor, auditory, somatosensory, and visual areas to describe the areal
expression patterns that we observed.
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Figure 1.
Hypotheses, predicted results, and interpretations
of analyses in this study. Diagrams are of dorsal views of the mouse
neocortex. A, Graded expression patterns of the
transcription factors Emx2, Pax6, and
Emx1 across the embryonic neocortex. Emx2
and Emx1 are expressed in a high caudomedial to low
rostrolateral gradient, whereas Pax6 is expressed in an
opposing gradient. B, Arrows indicate the
direction of the predicted shifts in markers of area identity in
Emx2, Pax6 (Sey/Sey), and
Emx1 loss-of-function mutants, if these genes are
involved in regulating arealization of the neocortex. The predicted
shifts are observed in Emx2 and Pax6
mutants but not in Emx1 mutants (indicated by red
X marks). C, Organization of the mouse
neocortex into areas predicted by our findings. These diagrams are not
intended to show the exact sizes and shapes of the primary neocortical
areas but rather to depict the disproportionate changes in area size
and positioning, or no changes, in arealization in the different
mutants. These predicted organizations suggested by our analyses of
gene markers and area-specific thalamocortical projections are limited
because the Emx2 and Pax6 mutants die on
the day of birth, before areas become anatomically and functionally
distinct, and thalamocortical projections do not develop in
Pax6 mutants. For simplicity, only the primary visual
(V1), motor (M1), and somatosensory
(S1) areas are shown. C, Caudal;
L, lateral; M, medial; R,
rostral; Sey, small eye mutant.
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Genes with expression patterns restricted to one area have yet to be
identified (Liu et al., 2000), but the layer-specific expression
domains of several of the genes examined here approximate the locations
of cortical areas or boundaries between them; citations are provided
for instances in which these relationships have been corroborated in
previous studies. However, even genes with restricted or graded
expression patterns that do not directly relate to a specific cortical
area or a boundary between areas can be used as markers for
position-dependent molecular properties of neocortical cells that
differ across the tangential or "areal" extent of the neocortex and
presumably are part of a repertoire of molecular markers that define
the area identity and characteristics of neocortical cells. Expansions
and contractions of the expression domains in the mutant neocortex, in
particular if the changes are consistent across the set of markers, are
evidence for changes in the position-dependent molecular properties of
neocortical cells.
Areal patterns of gene expression in the neocortex are altered
in opposing manners in Emx2 and Pax6
mutants
In the first part of this study, we examine in Emx2 and
Pax6 mutant mice the expression of seven genes that in
wild-type mice are expressed in restricted or graded patterns that
relate to the organization of the neocortex into areas. To facilitate
this comparison, we have divided the seven genes into three groups (Cad8, Cad6, and Id2;
RZR and p75; ephrin-A5 and
EphA7) on the basis of how effectively their
expression patterns complement or corroborate one another. The
expression of these genes is also layer-specific, and for most of them,
their areal expression patterns differ between layers. Although
Emx2 mutants exhibit minor lamination defects (Mallamaci et
al., 2000b), at a qualitative level of analysis, the changes in areal
patterning of the marker genes appears to be consistent across layers;
it is difficult to comment on this issue in the Pax6 mutants
because of the significant defects in lamination (Caric et al.,
1997).
Emx2 and Pax6 mutants:
Cad8, Cad6, and Id2
As we showed recently using whole-mount in situ
analysis (Bishop et al., 2000), the rostral domain of Cad8
expression observed in wild-type cortex exhibits a caudal expansion in
Emx2 mutants and a rostral restriction in Pax6
mutants (Fig.
2A-D).
However, these whole-mount in situ analyses of the cortical
hemisphere reveal only the superficial rostral expression domain of
Cad8, which corresponds to the high level of expression in
layers 2/3 of the motor area (Suzuki et al., 1997; Inoue et al., 1998).
Therefore, we have confirmed and extended the finding of Bishop et al.
(2000) by doing an analysis of the more complex, and revealing,
expression pattern of Cad8 obtained by in situ
hybridization on sections from E18.5 wild-type and mutant cortex.
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Figure 2.
Opposing changes in the expression domains of the
cadherin, Cad8 in Emx2, and
Pax6 (Sey/Sey) mutants.
A-D, Dorsal views of whole mounts of P0
cortical hemisphere of Emx2 wild-type (+/+)
(A), Emx2 mutant (/)
(B), Pax6 wild-type (+/+)
(C), and Pax6
(Sey/Sey) mutant (D) processed for
in situ hybridization using digoxygenin-labeled
riboprobes for Cad8. Arrowheads mark the
caudal limit of the rostral expression domain of
Cad8 in the superficial layers, which is characteristic of
motor areas. A'-D', Sagittal sections
through E18.5 brains of mice of the corresponding genotypes as in
A-D, processed for in
situ hybridization using S35-labeled
riboprobes for Cad8. Sections were later counterstained
with bisbenzimide. Sections are taken from similar mediolateral
positions; rostral is to the left and dorsal is to the
top. Each panel is a montage of
single-exposure photos using dark-field illumination with a red filter
to view the silver grains and with UV fluorescence to view the
counterstain. Marked are the approximate locations of the motor
(M), somatosensory
(S), and visual (V)
areas in the wild-type cortex and their shifted locations in the
Emx2/ cortex suggested by the expansion and caudal
shift in patterns of Cad8 expression, which are unique
in each of these areas in wild-type mice. Arrowheads in
A'-C' mark rostral and caudal expression
domains in the superficial layers characteristic of motor and visual
areas; in comparison, expression is substantially diminished in the
superficial layers of the intervening somatosensory area.
Arrows in A'-C' mark the
presumed border between motor and somatosensory areas. The
Cad8 expression rostral to these arrows
is the expression that is evident in the whole mounts shown in
A-C. This superficial rostral expression
domain characteristic of motor areas is essentially absent in
Pax6 (Sey/Sey) mutants (D,
D'). The wild type and mutants in each pair are
age-matched littermates. See Results for details. C,
Caudal; L, lateral; R, rostral.
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In wild-type mice, Cad8 is broadly expressed across the
neocortex within layer 5; however, throughout rostral neocortex,
Cad8 is also highly expressed in layers 2/3 (Suzuki et al.,
1997; Nakagawa et al., 1999) (Fig.
2A',C'). The caudal boundary of this
superficial expression domain (i.e., layers 2/3) has been reported to
correspond to the boundary between motor and somatosensory areas
(Suzuki et al., 1997). In Emx2 mutants, this superficial
rostral expression domain is expanded, and its caudal boundary is
shifted caudally (Fig. 2, compare A', B'). An
opposite and dramatic change is observed in Pax6 mutants in
which the superficial rostral expression domain is absent (Fig. 2,
compare C', D').
Similar to its rostral superficial expression domain, in caudal
neocortex corresponding to the visual area, Cad8 is also
expressed in layers 2/3 and deep to them, whereas in contrast,
expression in the somatosensory area positioned between the motor and
visual areas is very low in the superficial layers and limited mainly to layer 5 (Suzuki et al., 1997; Nakagawa et al., 1999) (Fig. 2A',C'). In Emx2 mutants, the
expression pattern characteristic of the somatosensory area also shifts
caudally and is found within caudal neocortex in the location normally
occupied by the caudal superficial Cad8 expression domain
characteristic of the visual area, which appears to be absent (Fig.
2B').
Figure 3 illustrates the expression of
Id2 and Cad6 in Emx2 and
Pax6 mutants and their wild-type littermates. In wild-type mice, both genes are broadly expressed across the neocortex, but each
exhibits differential laminar patterns of expression characterized by
abrupt changes in expression that are layer specific. Id2
exhibits strong expression in layer 5 in intermediate and caudal parts of the neocortex but weak expression in rostral parts. The change in
layer 5 from strong to weak expression occurs abruptly at a position
that corresponds to the boundary between motor and somatosensory areas
(Rubenstein et al., 1999). This boundary between strong and weak layer
5 expression is shifted caudally in Emx2 mutants and
rostrally in Pax6 mutants. In addition, wild-type mice
exhibit another differential expression pattern specific for layers
2/3. Id2 is strongly expressed in layers 2/3 of rostral
neocortex, but in intermediate parts of the neocortex (e.g., the
somatosensory area), the expression level declines rapidly to low or
nondetectable levels characteristic of caudal neocortex (i.e., the
visual area). The rostral domain of strong expression in layers 2/3
expands caudally in Emx2 mutants, leaving only the extreme
caudal pole of the cortical hemisphere with a low level of
Id2 expression. In Pax6 mutants, the domain of
strong expression characteristic of layers 2/3 of rostral neocortex
appears to be absent throughout the neocortex.
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Figure 3.
Id2 and Cad6, used
as markers of rostral neocortical areas, show opposing shifts in
Emx2 and Pax6 mutants. In
situ hybridizations on sagittal sections through the forebrain
of E18.5 mice using S35-labeled riboprobes for the
HLH transcription factor, Id2
(A-B') or the cadherin,
Cad6 (C-D'), and
counterstained with bisbenzimide are shown. Sections are from
Emx2 wild-type (+/+) and mutant (/) littermates or
Pax6 wild-type (+/+) and mutant (Sey/Sey)
littermates and are taken from similar medial-lateral positions. Each
panel is a montage of single-exposure photos using
dark-field illumination with a red filter to view the silver grains and
UV fluorescence to view the counterstain. Id2 exhibits a
graded expression in superficial layers of rostral areas in wild-type
mice; the arrows in A and A' mark the
position where the expression declines to very low levels. The
arrowheads in A-B' mark the
transition from low to high expression reported in layer 5; this
transition corresponds to the border between motor and somatosensory
areas. The asterisks in C, C', and
D mark a domain of low Cad6 expression normally
characteristic of far rostral neocortex. This domain of low expression
expands and shifts caudally in Emx2 mutants, whereas in
Pax6 mutants it shifts rostrally and essentially disappears
(C', D', long arrows). See Results for
details. C, Caudal; R, rostral.
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In wild-type mice, Cad6 is expressed prominently in dorsal
and lateral parts of the neocortex corresponding to somatosensory and
auditory areas and exhibits a domain of low expression, especially deficient in layers 4 and 5, in extreme rostral neocortex (Suzuki et
al., 1997; Nakagawa et al., 1999). In Emx2 mutants, the
domain of low Cad6 expression normally confined to extreme
rostral neocortex is greatly expanded and covers virtually the rostral
half of the neocortex. In contrast, this domain is essentially absent
in Pax6 mutants, and high levels of Cad6
expression continue to the rostral pole of the neocortex.
These data on changes in the expression of Cad8,
Cad6, and Id2 are consistent with an expansion of
rostral neocortical areas and a reduction of caudal areas in
Emx2 mutants and a contraction of rostral neocortical areas
and an expansion of caudal areas in Pax6 mutants.
Emx2 and Pax6 mutants:
RZR and p75
Figure 4 illustrates the expression
of RZR (ROR) and p75 at E18.5 in
Emx2 and Pax6 mutants and their wild-type
littermates. RZR and p75 exhibit strongly
graded patterns of expression across the neocortex, but in opposing
directions (Mackarehtschian et al., 1999; Rubenstein et al., 1999). In
wild-type mice, RZR exhibits a graded expression within
layer 4 that extends across most of the rostrocaudal extent of the
neocortex; expression is strong rostrally and progressively declines to
very low levels near the extreme caudal pole of the neocortex. In
addition to this reported graded expression within layer 4 (Rubenstein
et al., 1999), we find that RZR is also differentially
expressed within layer 5, with a domain of high expression restricted
to the rostral pole of the neocortex that exhibits a rapid decline of
expression to reach very low levels of expression at about the midpoint
of the neocortex; expression is very low or nondetectable in the caudal half of the neocortex.
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Figure 4.
The complementary expression patterns of
RZR and p75 show opposing shifts in
Emx2 and Pax6 mutants. In
situ hybridizations on sagittal sections through the forebrain
of E18.5 mice using S35-labeled riboprobes for
either the nuclear receptor RZR
(A-B') or the neurotrophin receptor
p75 (C-D') and
counterstained with bisbenzimide are shown. Sections are from
Emx2 wild-type (+/+) and mutant (/) littermates or
Pax6 wild-type (+/+) and mutant (Sey/Sey)
littermates and are taken from similar medial-lateral positions. Each
panel is a montage of single-exposure photos using
dark-field illumination with a red filter to view the silver grains and
UV fluorescence to view the counterstain. RZR shows
two distinct high rostral to low caudal gradients across the wild-type
neocortex (A, B), a gradient in
superficial layers that extends farther caudally (marked by
arrowheads) than a gradient within the deeper layers
(marked by short arrows). Both gradients of
RZR expression expand caudally in Emx2
mutants (A') and constrict rostrally in
Pax6 mutants (B'). p75 is
expressed in the deep layers in roughly the caudal half of the
wild-type neocortex (C, D).
p75 expression constricts caudally in
Emx2 mutants (C') and expands rostrally
in Pax6 mutants (D'). The
arrowheads mark the rostral limit of expression. The
long arrows in A'-D' indicate
the opposing shifts in expression in the mutants. See Results for
details. C, Caudal; R, rostral.
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Each of these layer-specific graded expression patterns of
RZR exhibit opposing changes in Emx2 and
Pax6 mutants. In Emx2 mutants, RZR
is highly expressed in layer 4 across virtually the entire neocortex,
and the domain of RZR layer 5 expression characteristic
of rostral neocortex is substantially expanded and exhibits high
expression throughout intermediate parts of the neocortex and well into
caudal neocortex. In Pax6 mutants, we observe the opposite
changes in expression, with virtually no expression detected in any
layers in the caudal half of the neocortex.
In wild-type neocortex, p75 expression is confined to the
subplate and layer 6 of caudal neocortex and tapers off sharply to
nondetectable levels of expression rostrally (Mackarehtschian et al.,
1999). In Emx2 mutants, the domain of p75
expression is contracted and shifted caudally. The changes in
p75 expression are more impressive in the Pax6
mutants, in which the domain of p75 expression expands
rostrally to such an extent that it covers virtually the entire
rostrocaudal axis of the neocortex.
These data on changes in the expression of RZR and
p75 are consistent with an expansion of rostral neocortical
areas and reduction of caudal areas in Emx2 mutants and
contraction of rostral neocortical areas and expansion of caudal areas
in Pax6 mutants.
Emx2 and Pax6 mutants:
ephrin-A5 and EphA7
Figure 5 illustrates the expression
of ephrin-A5 and EphA7 at E18.5 in
Emx2 and Pax6 mutants and their wild-type
littermates. In wild-type mice, ephrin-A5 is expressed most
highly in dorsolateral parts of the neocortex that correspond to the
somatosensory area (Mackarehtschian et al., 1999). The domain of high
ephrin-A5 expression is shifted and restricted caudally in
Emx2 mutant neocortex, whereas in Pax6 mutants,
the domain of high ephrin-A5 expression is shifted and
restricted rostrally. In wild-type mice, EphA7 is expressed throughout the neocortex but at much lower levels in intermediate parts
of the neocortex corresponding to the somatosensory area (Rubenstein et
al., 1999). In Emx2 mutants, this domain of lower EphA7 expression is reduced in extent and shifted caudally,
whereas in Pax6 mutants a domain of lower EphA7
expression similar to that observed in wild type is not evident, but a
small domain of reduced expression is apparent and restricted to a more
rostral position. These data on changes in the expression of
ephrin-A5 and EphA7 are consistent with an
expansion of rostral neocortical areas and reduction of caudal areas in
Emx2 mutants and contraction of rostral neocortical areas
and expansion of caudal areas in Pax6 mutants.
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Figure 5.
The complementary expression patterns of
ephrin-A5 and EphA7, used as markers of
intermediate neocortical areas, show opposing shifts in
Emx2 and Pax6 mutants. In
situ hybridizations on sagittal sections through the forebrain
of E18.5 mice using S35-labeled riboprobes for the
axon guidance ligand, ephrin-A5, and one of its
receptors, EphA7, and counterstained with bisbenzimide
are shown. Sections are from Emx2 wild-type (+/+) and
mutant (/) littermates (A-B') or
Pax6 wild-type (+/+) and mutant (Sey/Sey)
littermates (C-D') and are taken from
similar medial-lateral positions. Each panel is a
montage of single-exposure photos using dark-field illumination with a
red filter to view the silver grains and UV fluorescence to view the
counterstain. In wild-type mice, ephrin-A5 has high
expression centered on the somatosensory area (A,
B), whereas EphA7 has low expression
centered on the somatosensory area (C,
D). These domains shift caudally in Emx2
mutants (A'-B') and rostrally in
Pax6 mutants (C'-D').
Arrowheads mark the domains of high
ephrin-A5 or low EphA7 expression. See
Results for details. C, Caudal; R,
rostral.
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Areal patterns of gene expression in the neocortex of
Emx1 mutants resemble those in wild-type mice
The changes in the patterns of gene markers described
above indicate that EMX2 and PAX6 are involved in regulating
the areal patterning of molecular correlates of neocortical
arealization. In the second part of this study, we have performed a set
of analyses to determine whether EMX1 may also confer
position-dependent or area-specific properties to neocortical cells.
First, we have used the same panel of seven genes as markers for
potential changes in arealization of Emx1 mutant neocortex
by doing a comparison of gene expression patterns in
Emx1+/+, +/, and / littermates. As illustrated in
Figures 6 and
7, we observe no significant alterations in the areal expression patterns of the panel of seven genes analyzed (data from the Emx1 heterozygous mice are not shown).
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Figure 6.
Patterned expression of
Cad8 and Cad6 appears normal in
Emx1 mutants. A-D, Dorsal
views of whole mounts of E18.5 cortical hemispheres of
Emx1 wild-type (+/+) and mutant (/) mice processed
for in situ hybridization using digoxygenin-labeled
riboprobes for Cad8 (A, B)
or Cad6 (C, D).
Arrowheads in A and B mark
the caudal limit of the rostral Cad8 expression domain
characteristic of motor areas and mark the lateral expression domain of
Cad6 in C and D.
A', B', Sagittal sections through E18.5
brains of Emx1 wild-type (+/+) and mutant (/) mice
processed for in situ hybridization using
S35-labeled riboprobes for Cad8.
Sections were later counterstained with bisbenzimide. Sections are
taken from similar medial-lateral positions; rostral is to the
left and dorsal is to the top. Each
panel is a montage of single-exposure photos using
dark-field illumination with a red filter to view the silver grains and
UV fluorescence to view the counterstain. Marked are the approximate
locations of the motor (M),
somatosensory (S), and visual
(V) areas suggested by the patterns of
Cad8 expression, which are unique in each of these areas
in wild-type mice. Arrowheads in
A'-B' mark rostral and caudal expression
domains in the superficial layers characteristic of motor and visual
areas; in comparison, expression is substantially diminished in the
superficial layers of the intervening somatosensory area. Arrows in
A'-B' mark the presumed border between
motor and somatosensory areas. The Cad8 expression
rostral to these arrows is the expression that is
evident in the whole mounts shown in A and
B. The wild-type and mutants in each pair are
age-matched littermates. See Results for details. C,
Caudal; L, lateral; R, rostral.
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Figure 7.
The expression domains
of genes that mark rostral, caudal, or intermediate areas of the
neocortex appear normal in Emx1 mutants. In
situ hybridizations on sagittal sections through the forebrain
of E18.5 Emx1 wild-type (+/+) and mutant (/)
littermates using S35-labeled riboprobes for
Id2, RZR, p75,
ephrin-A5, and EphA7 and counterstained
with bisbenzimide are shown. Sections are taken from similar
medial-lateral positions. Each panel is a montage of
single-exposure photos using dark-field illumination using a red filter
to view the silver grains and UV fluorescence to view the counterstain.
Id2 exhibits a graded expression in superficial layers
of rostral areas in wild-type mice; the arrows in
A and A' mark the position where the
expression declines to very low levels. The arrowheads
in A-B' mark the transition from low to
high expression reported in layer 5; this transition corresponds to the
border between motor and somatosensory areas. RZR is
expressed in two distinct high rostral to low caudal gradients across
the neocortex (B, B'); a gradient in
superficial layers that extends farther caudally (marked by
arrowheads) than a gradient within the deeper layers
(marked by short arrows). p75 is
expressed in the deep layers in roughly the caudal half of the
neocortex (C, C'). The
arrowheads mark the rostral limit of expression.
ephrin-A5 has high expression centered on the
somatosensory area (D, D'), whereas
EphA7 has low expression centered on the somatosensory
area (E, E'). Arrowheads
mark the domains of high ephrin-A5 or low
EphA7 expression. Overall, the areal expression patterns
of this panel of marker genes are similar in wild-type and mutant
neocortex. See Results for details. C, Caudal;
R, rostral.
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Expression analysis of Cad8 and Cad6 was
done using in situ hybridization on both whole mounts and
sagittal sections of E18.5 in wild-type and Emx1 mutants
(Fig. 6). In whole mounts of wild-type brains, the expression domain of
Cad8 is restricted to rostral neocortex, which corresponds
to the high level of expression in layers 2/3 of the motor area (Suzuki
et al., 1997; Inoue et al., 1998). This rostral expression domain of
Cad8 is unaltered in Emx1 mutants compared with
wild type (Fig. 6A,B). In whole
mounts of wild-type brains, Cad6 is expressed in a domain in
lateral neocortex corresponding to somatosensory and auditory areas
(Suzuki et al., 1997; Inoue et al., 1998). This expression domain of
Cad6 appears unaltered in Emx1 mutants
compared with wild type (Fig. 6C,D). Expression
analyses show that the more complex expression patterns observed in
sagittal sections are also similar between wild-type and
Emx1 mutants for both Cad8 (Fig.
6A',B') and Cad6 (data not
shown). Expression analyses of Id2, RZR,
p75, ephrin-A5, and EphA7, on sagittal
sections also reveal very similar patterns in the cortex of
Emx1 mutants compared with wild type (Fig. 7). Taken
together, these findings indicate that molecular markers of neocortical
areas are expressed normally in Emx1 mutant neocortex.
Area-specific thalamocortical projections appear normal in
Emx1 mutants
As an additional assessment of the potential role for
EMX1 in regulating neocortical area identity, we
used DiI and DiA as retrograde axon tracers to assess area-specific
thalamocortical projections in newborn Emx1 mutant mice and
their wild-type littermates. In wild-type mice, placements of DiI
crystals confined to the cortical plate of occipital cortex, the
location of the primary visual area, retrogradely label neurons in the
dLG (Fig. 8A). Placement of DiI crystals at the same site in Emx1
mutants results in a distribution of retrogradely labeled neurons (Fig.
8B) similar to that observed in wild type (Fig.
8A). In wild-type mice, placements of DiA crystals
confined to the cortical plate of parietal cortex, the location of the
somatosensory area, retrogradely label neurons in the ventroposterior
thalamic nucleus (Fig. 8A', VP). Placement of DiA crystals at the same site in Emx1 mutants results in
a distribution of retrogradely labeled neurons (Fig.
8B') similar to that observed in wild-type mice (Fig.
8A'). Thus, retrograde labeling from somatosensory
and visual areas of the neocortex indicates that thalamocortical
connections in Emx1 mutants exhibit normal area-specific
patterns of connections. Taken together, our analyses using molecular
markers and axon tracing suggest that the Emx1 null mutation
does not affect arealization in embryonic or neonatal mice.
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Figure 8.
Area-specific thalamocortical projections appear
normal in Emx1 mutant mice. Sagittal sections through P0
Emx1 wild type (+/+) and mutant (/) brains showing
retrograde DiI (red) and DiA
(green-bluish) labeling and bisbenzimide
counterstain (dark blue). Rostral is to the
left and dorsal is to the top in all
panels. A, B, An injection
of DiI into visual (occipital) cortex retrogradely labels neurons in
the dorsal lateral geniculate nucleus (dLG) in both
Emx1+/+ and Emx1/ mice.
C, D, An injection of DiA into
somatosensory (parietal) cortex of the same set of brains retrogradely
labels neurons in the ventroposterior thalamic nucleus
(VP) in both Emx1+/+ and
Emx1/ mice. Each pair of sections is from the same
brain; the dLG-labeled sections are lateral to those with the VP
labeling. See Results for details.
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Areal patterns of gene expression in the neocortex of
Emx double mutants resemble those in
Emx2 single mutants
The coincident graded expression of Emx1 and
Emx2 in the cortical ventricular zone, and their high
sequence homology, suggests the possibility that the lack of an
arealization phenotype in the Emx1 mutants may be caused by
Emx2 compensating for the loss of Emx1. To
investigate this possibility, we examined the expression patterns of
the panel of seven gene markers in sagittal sections through
Emx1/; Emx2/ double mutant and wild-type (i.e.,
Emx1+/; Emx2+/+)
littermates at E18.5. Because Emx double mutants lack a TCA projection (K. M. Bishop, S. Garel, J. L. R. Rubenstein, and D. D. M. O'Leary, unpublished
observations), we were not able to examine the areal patterning of this projection.
In Figure 9, we illustrate the expression
patterns for four of the seven genes analyzed (Cad6,
p75, ephrin-A5, and EphA7), and
for ease of comparison, we include panels showing the expression of
these genes in Emx2 single mutant neocortex. The findings
from these four markers are representative of the findings from the full panel of seven markers. In the areal dimension, the expression domains of each of these markers are shifted caudally in Emx1; Emx2 double mutants compared with wild type. Because the
tangential extent of the neocortex is substantially reduced in the
Emx1; Emx2 double mutants compared with wild-type mice (by
~50% in its surface area), one needs to be somewhat cautious in
interpreting patterns of gene expression. However, the relative shift
and positioning of the expression domain of each of these marker genes
in the Emx1; Emx2 double mutants appears to be similar to
that in the Emx2 single mutants. Thus, the lack of a
defective arealization phenotype in Emx1 mutants is not
caused by Emx2 compensating for the loss of Emx1.
Therefore, these findings support the conclusion from our analyses of
the Emx1 mutants that EMX1 has no apparent role in
regulating arealization of the neocortex.
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Figure 9.
The relative positioning of expression domains of
gene markers in Emx1; Emx2 double mutant neocortex
resembles that in Emx2 single mutant neocortex. Shown
are in situ hybridizations on sagittal sections through
the forebrain of E18.5 (A-D) wild-type (i.e.,
Emx1+/; Emx2 +/+)
(A'-D') Emx double mutant
(Emx1/; Emx2/), and
(A''-D'') Emx2 single
mutant (Emx2/) mice using
S35-labeled riboprobes for Cad6,
p75, ephrin-A5, or EphA7
and later counterstained with bisbenzimide. Note that the neocortex of
the Emx double mutant is reduced to approximately half of the
wild-type area. Sections are taken from similar medial-lateral
positions. Each panel is a montage of single-exposure
photos using dark-field illumination and a red filter to view the
silver grains and UV fluorescence to view the counterstain. The
asterisks in A-A'' mark a
domain of low Cad6 expression normally characteristic of
far rostral neocortex. This domain of low expression expands and shifts
caudally in Emx double mutants and Emx2 mutants.
p75 is expressed in the deep layers in roughly the
caudal half of the wild-type neocortex (B); this
expression domain constricts caudally in Emx double mutants and
Emx2 mutants (B', B'').
The arrowheads mark the rostral limit of expression. In
wild-type mice, ephrin-A5 has high expression centered
on the somatosensory area (A, B), whereas
EphA7 has low expression centered on the somatosensory
area (C, D). These domains shift caudally
in Emx double mutants and Emx2 mutants
(C', D', C'',
D''). Arrowheads mark the domains of high
ephrin-A5 or low EphA7 expression. See
Results for details. C, Caudal; R,
rostral.
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DISCUSSION |
Defining roles for Emx1, Emx2, and
Pax6 in arealization
We have presented evidence that strengthens the claim that
EMX2 and PAX6 regulate arealization of the developing
neocortex and confer positional identities to cortical cells. In
addition, we show that EMX1 has no apparent role in regulating
arealization. Figure 1 summarizes our predictions and interpretations.
Because Emx2 and Pax6 (Sey/Sey) mutant
mice die on the day of birth before areas become anatomically and
functionally distinct, we have used genetic markers to assess changes
in positional, or area, identity of cortical cells and presumably
associated changes in arealization of the neocortex. We are confident
in basing our interpretations on these data because each of the seven
marker genes analyzed exhibits opposing expansions or contractions, and
border shifts, in its expression domain in Emx2 and
Pax6 mutants, as predicted by the opposing gradients of
Emx2 and Pax6 expression (Fig. 1). The changes in
marker expression indicate that arealization of the neocortex is
disproportionately altered in Emx2 and Pax6
mutant mice in opposing manners: in Emx2 mutants,
rostrolateral areas are expanded compared with wild type, whereas
caudomedial areas are reduced, and in Pax6 mutants the
opposite changes occur. We conclude that EMX2 and PAX6 act in
opposing manners to regulate arealization and to confer positional, or
area, identity to neocortical cells. EMX2 appears to
preferentially impart caudomedial area identities, and PAX6
preferentially imparts rostrolateral area identities.
We expected to observe similar changes in neocortical arealization in
Emx1 mutants as in Emx2 mutants, because in the
ventricular zone, Emx1 has a graded expression similar to
that of Emx2 (Fig. 1). However, we find that each of the
seven marker genes exhibits a normal expression pattern in the
neocortex of Emx1 mutants. In addition, the area-specific
organization of TCA projections is normal in Emx1 mutants.
These findings suggest that unlike EMX2, EMX1 is not involved
in arealization. An alternative is that Emx function is redundant in
arealization and that EMX2 in particular can more fully compensate
for the loss of EMX1 than vice versa. This is suggested by the
coincident expression of the Emx genes in the cortical ventricular
zone, their high sequence homology, and that in the dorsal
telencephalon Emx2 expression begins at E8.0-8.5, whereas
Emx1 expression begins at E9.5 (Simeone et al., 1992).
However, our marker analysis of Emx1; Emx2 double mutants
reveals that the changes in patterns of gene expression are similar to
those observed in Emx2 single mutants. Thus, unlike EMX2, EMX1 does not appear to play a role in regulating
arealization of the neocortex.
Emx genes may function in the patterning of neocortical areas in
the same way that their Drosophila ortholog, the empty
spiracle (ems) gene, specifies in the developing
head early populations of neuroblasts that at later stages form
specific brain structures (Younossi-Hartenstein et al., 1997; Hartmann
et al., 2000). If the Emx genes act in this manner, we would predict
that a deletion of both would result in the complete loss of
caudomedial neocortical tissue, along with areas that differentiate
within it, such as visual areas. However, our findings are inconsistent
with this scenario, because rather than a complete loss of tissue in
which caudal areas would form, our marker analyses indicate that
rostral neocortical areas expand and shift caudally into domains that would normally develop caudal area identities and that caudal areas are
present but contracted. It seems more likely that EMX2 confers
positional identity to cortical neurons around the time they are
generated. Further support for this interpretation is our finding that
the patterned expression of the marker genes and the positioning of
their expression domains retain the same relative relationships
with each other, and with the neocortex as a whole, between
Emx2 single mutants and Emx1; Emx2 double mutants, although the double mutant cortex is approximately half the
normal size.
Because surface area of the cortex is reduced by ~25% in
Emx2 mutants and Pax6 mutants (Bishop et al.,
2000), the disproportionate areal expansions and contractions in these
mutants may be caused in part by a decreased growth of caudomedial
cortex in Emx2 mutants and rostrolateral cortex in
Pax6 mutants (Muzio et al., 2002). However, several aspects
of our findings cannot be accounted for by a regional decrease in
cortical growth and therefore strongly argue that positional identity
and arealization of the neocortex are altered in Emx2 and
Pax6 mutants (Bishop et al., 2000; present study). As
discussed above, these include the similarities in changes in
expression domains of areal markers between Emx2 single and
Emx1; Emx2 double mutants, although cortical size in the
double mutant is substantially decreased compared with the single
mutant. Other aspects of our findings supporting this argument include that the expression domains of areal markers exhibit the following: (1)
increases or decreases in their proportional size relative to that of
the neocortex which significantly exceed that predicted by a
region-specific decrease in growth, and for some markers, even
increases in absolute size of domains of positive or negative expression, and (2) changes in relative positioning and coverage of the
rostrocaudal axis of the neocortex that cannot be accounted for by a
region-specific decrease in growth.
Potential mechanisms for EMX2 and PAX6 regulation
of arealization
The details of how PAX6 and EMX2 regulate arealization are
unknown. The loss of one or the other may result in a complete change
or switch in area identity. PAX6, for example, has been implicated more
generally in regulating the identity of ventrolateral regions of the
cerebral cortex. In Pax6 mutants, progenitor cells in the
ventrolateral regions lose expression of cortical regulatory genes and
acquire expression of subcortical regulatory genes. As a result,
ventral cortical regions (e.g., olfactory cortex) are hypoplastic,
whereas adjacent subcortical areas expand (Stoykova et al., 2000;
Toresson et al., 2000; Kim et al., 2001; Yun et al., 2001; Muzio et
al., 2002). Evidence for cross-repression between EMX2 and PAX6 in the
neocortex (Muzio et al., 2002) suggests that they may cooperate with
each other, and possibly with other transcription factors (e.g., LHX2)
(Nakagawa et al., 1999; Monuki et al., 2001), to confer positional or
area identity to neocortical neurons. For example, EMX2 and PAX6 may
participate in a combinatorial code of regulatory proteins that
specifies the area identity of cortical neurons, as suggested for the
specification of subtypes of motor neurons and interneurons in the
spinal cord (Jessell, 2000). If so, the loss of either EMX2 or PAX6 may
perturb the area identity of cortical neurons, resulting in an aberrant
or chimera area identity, rather than a complete change from one area
identity to another.
One protein that may collaborate with EMX2 and PAX6 in regulating
arealization is COUP-TFI, an orphan nuclear receptor that has a high
caudal to low rostral graded expression across the neocortex within the
ventricular zone, subplate, and cortical plate (Liu et al., 2000). An
analysis in CoupTfI mutants of the expression of the marker
genes, Cad8, Id2, and ROR
(RZR), shows that they all lose their normally
restricted, areal expression patterns and instead are broadly expressed
across the neocortex (Zhou et al., 2001). This finding differs
from that in Emx2 and Pax6 mutants, in which the
marker genes retain specific patterns of expression, but the patterned
expression is either expanded or contracted in a manner that opposes
the graded expression of Emx2 or Pax6. In
contrast, in CoupTfI mutants, Emx2 and
Pax6 show their normal graded patterns of expression. Thus,
CoupTfI mutants have an apparent loss of areal specificity
in patterns of marker expression, with the exception of Emx2
and Pax6, suggesting that COUP-TFI does not directly
regulate arealization but is required for the proper action of EMX2 and
PAX6 in this process.
Establishment of graded expression of arealization genes across
the neocortex
Evidence is emerging for a role for secreted signaling proteins
with known patterning functions in establishing and maintaining the
graded expression of regulatory genes, in particular Emx2, across the neocortical ventricular zone. One such signaling protein is
FGF8, which is expressed in proximity to rostral parts of the cortical
anlage at neural plate stages, in the anterior neural ridge, and later
the rostrodorsal midline of the telencephalon (Crossley and Martin,
1995; Shimamura and Rubenstein, 1997; Crossley et al., 2001).
Electroporation in E11.5 mouse cortex of vectors to overexpress FGF8 or
a soluble FGF8 receptor body to diminish endogenous FGF8 results in
shifts in areal markers (Fukuchi-Shimogori and Grove, 2001) similar to
those expected if the graded expression of Emx2 was
decreased or increased. That these effects are likely caused by FGF8
regulation of Emx2 is implied by the finding that FGF8-soaked beads implanted into dorsal telencephalon of embryonic chicks locally repress Emx2 expression (Crossley et al.,
2001).
Other candidate regulators of Emx2 expression include
members of the bone morphogenetic protein family, which along with
Wnt family members are expressed in the cortical hem, a caudal
midline structure adjacent to the hippocampal anlage (Furuta et al.,
1997, Grove et al., 1998). Ectopic expression of Bmp4 in the
dorsal telencephalon of embryonic chicks appears to enhance
Emx2 expression, either directly or through the repression
of Fgf8 (Ohkubo et al., 2002). The zinc finger transcription
factor Gli3, which is expressed broadly in the dorsal
telencephalon, may be upstream to both Emx genes, because the
expression of Emx1 is lost (Thiel et al., 1999; Tole et al.,
2000), and Emx2 is lost (Thiel et al., 1999) or reduced (Tole et al., 2000) in the cortex of the
extra-toesJ
(XtJ) mutant mouse, a naturally occurring
Gli3 mutant (Franz, 1994). In contrast, Pax6
expression is maintained in the XtJ mutant
(Thiel et al., 1999; Tole et al., 2000). However, it is unclear
whether Emx2 is normally regulated directly by GLI3, by signaling molecules produced in the cortical hem that are lost in the
Xt mutant, or by other molecular deficiencies in
XtJ mutants (Grove et al., 1998;
Thiel et al., 1999; Tole et al., 2000).
Both PAX6 and EMX2 are implicated in regulating members of
the Wnt family, which in turn may influence corticogenesis. In Pax6 mutants, expression of Wnt7b and
SFRP2 in ventrolateral cortical progenitors is reduced
(Kim et al., 2001), whereas Wnt3a and
Wnt8b expression in the dorsomedial cortex is expanded
(Muzio et al., 2002). On the other hand, Wnt3a expression is
reduced in dorsomedial cortex in Emx2 mutants (Muzio et al.,
2002).
Defining area identity at the level of patterned
gene expression
Many genes, including markers used in this study, exhibit abrupt
transitions in their expression patterns within the cortical plate, and
the areas themselves usually have abrupt borders. Therefore, the graded
expressions of Emx2 and Pax6 are likely
translated to generate downstream gene expression in restricted
patterns with abrupt borders. Studies in Drosophila embryos
have defined distinct mechanisms through which graded regulatory
proteins can generate sharply bordered patterns of downstream gene
expression, for example through concentration-dependent differences in
binding efficacy to promoter and repressor elements (Rusch and Levine, 1996) or the combinatorial action of multiple activators and repressors of transcription (Stanojevic et al., 1991; Small et al., 1996). In the
developing spinal cord, sonic hedgehog secreted by the notocord and
floorplate represses or induces in the ventricular zone the expression
of different classes of transcription factors in graded patterns, which
are progressively converted into sharply bordered patterns through
mutual repression (Jessell, 2000). This mechanism results in
genetically distinct domains of progenitors, which generate different
subtypes of spinal interneurons and motor neurons definable by their
expression of unique subsets of transcription factors.
Mechanisms similar to those used in spinal cord are likely coopted to
generate neocortical areas and area-specific identities of neurons, but
some differences appear to exist. For example, at no time during
corticogenesis are sharply bordered patterns of regulatory genes
observed in the ventricular zone; all retain graded expression
patterns. Initially, even genes differentially expressed in the
cortical plate have graded patterns, although at later stages of
development many acquire an expression pattern with abrupt changes that
correlate with borders between areas (Rubenstein et al., 1999; Sestan
et al., 2001).
Area specificity may differ among layers
Genes with an expression pattern restricted to one area have not
been identified (Liu et al., 2000). The only genetic marker with an
expression pattern restricted to one area is the H-2Z1 transgene, which
marks the granular parts of postnatal mouse S1 (Cohen-Tannoudji et al.,
1994). It seems reasonable to conclude then that a neocortical area and
the area identity of the neurons that comprise it are defined by the
expression of a unique subset of genes, each of which is also expressed
in other areas. However, the actual scenario is more complex
because each layer has a unique profile of gene expression: most genes
reported to be differentially expressed in the neocortex and expressed
in more than one layer, including markers that we use here, have
different expression patterns in each layer. The expression of
Id2 and RZR are clear examples described in
this study (see Results). Thus, although neurons in different layers
are generated by the same progenitors (Monuki and Walsh, 2001), they
appear to have distinct positional or area identities, at least in
terms of their expression of genes used as markers of these identities.
This feature has significant implications for the genetic regulation of
areas and how area identity is encoded in the ventricular zone and
imparted by progenitors to their progeny. An understanding of these
mechanisms will require the definition of areas at the level of gene
expression, and defining the relationship between the specification of
layer-specific and area-specific properties.
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FOOTNOTES |
Received March 11, 2002; revised June 17, 2002; accepted June 17, 2002.
This work was supported by National Institute of Neurological Disorders
and Stroke Grants NS31558 (D.O.) and NS34661 (J.R.), a McKnight
Investigator Award (D.O.), National Institute of Mental Health Grant
K02 MH01046 (J.R.), and postdoctoral fellowships from the Natural
Sciences and Engineering Research Council of Canada and the Canadian
Institutes for Health Research (K.B.). We are grateful to P. Gruss for
breeding pairs of Emx2 mutant mice, M. Goulding for
Sey mice, and various investigators for plasmids (see
Materials and Methods). We thank Y. Nakagawa and N. Dwyer for helpful
discussions and comments.
Correspondence should be addressed to Dr. Dennis D. M. O'Leary,
Molecular Neurobiology Laboratory, The Salk Institute for Biological
Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037. E-mail:
doleary{at}salk.edu.
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REFERENCES |
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ABSTRACT
INTRODUCTION
